Unconventional PDZ Recognition Revealed in α7 nAChR-PICK1 Complexes

PDZ domains are modular domains that conventionally bind to C terminal or internal motifs of target proteins to control cellular functions through the regulation of protein complex assemblies. Almost all reported structures of PDZ-target protein complexes rely on fragments or peptides as target proteins. No intact target protein complexed with PDZ was structurally characterized. In this study, we used NMR spectroscopy and other biochemistry and biophysics tools to uncover insights into structural coupling between the PDZ domain of protein interacting with C-kinase 1 (PICK1) and α7 nicotinic acetylcholine receptors (α7 nAChR). Notably, the intracellular domains of both α7 nAChR and PICK1 PDZ exhibit a high degree of plasticity in their coupling. Specifically, the MA helix of α7 nAChR interacts with residues lining the canonical binding site of the PICK1 PDZ, while flexible loops also engage in protein–protein interactions. Both hydrophobic and electrostatic interactions mediate the coupling. Overall, the resulting structure of the α7 nAChR-PICK1 complex reveals an unconventional PDZ binding mode, significantly expanding the repertoire of functionally important PDZ interactions.

. Overlay of 2D saturation transfer (STD) NMR spectra of the α7nAChR TMD+ICD without (purple) and with (green) saturation of the PICK1 I33 amide proton (~11.6 ppm).STD spectra were collected in an interleaved fashion with off-and on-resonance saturation of 1 HN peak (~11.6 ppm) of I33 for 0.5s saturation and a recycle time of 1.5s.The off-resonance frequency was set at 20 ppm, which is far away from the 1 H frequencies of both the 15 N-labeled α7nAChR TMD+ICD and the unlabeled PICK1 PDZ domain.The selective saturation was achieved using an IBURP2 pulse train (50 ms Gaus1.1000-shaped with an inter-pulse delay of 4 μs).The spectra were acquired at 800 MHz NMR spectrometer at 45 ºC.The paramagnetic condition resulted from the MTSL labeling at residues C44 and C46 of the PDZ.
The diamagnetic condition was introduced by ascorbic acid (2.5 mM).Several inserts are provided to assist visualization of signal changes under the paramagnetic and diamagnetic conditions.These spectra were acquired at 800 MHz NMR spectrometer at 35 ºC.(a) Sequence alignment of MA helices of the human α7, α4, and β2 nAChRs.The α7 residues colored in orange are closely interacting with the PDZ domain in the α7nAChR-PICK1 complex structural model.To validate the complex structure and assess how mutations in the α7 MA helix influence the α7nAChR-PICK1 complex formation, we designed an α7 mutant of three mutations (N423D, E430D and S431D), named α7DDD, to match these three α7 residues with the equivalent residues in α4 or/and b2, because neither α4 nor b2 was found binding PICK1 previously. 1 (b) Overlay of size exclusive chromatography (SEC) profiles of the purified wild type (α7WT, black) and mutant (α7DDD, red) α7nAChR.The same SEC profile and peak position (~1.4 ml) of α7WT and α7DDD suggests that α7DDD retains the pentameric assembly as α7WT.The SEC was performed using a Superdex 200 Increase 3.2/300 column with 10 mM phosphate buffer at pH 7.8, 150 mM NaCl and 0.05% LDAO.(c) 15% SDS-PAGE gel showing pulldown of the PICK1 PDZ domain by α7WT and α7DDD with their his-tags as baits.Comparing to α7WT, α7DDD shows a ~50% decrease in pulldown of PICK1 based on band intensity ratios of PICK1/α7WT vs. PICK1/α7DDD.The slower migration of α7DDD in the SDS-PAGE results from an increase of the net negative charge in α7DDD as reported previously. 2 The experiment was carried out following the protocol described in the Method section.

Figure S1 .
Figure S1.Impact of the α7nAChR MA helix deletion to binding PICK1.Intensity ratios (PICK1/α7nAChR) are obtained from measuring individual band intensities in each lane of the SDS-page resulted from pulldown experiments (Fig. 2).Relative to the full length α7nAChR (α7WT), deletion of MA residues reduced PICK1 pulldown.α7Δ419-423 in the middle of the MA helix decreased PICK1 pulldown by ~53%.α7Δ428-432 toward top of the MA helix decreased PICK1 pulldown by ~86%.Interestingly, α7Δ408-432, a much more extended deletion, resulted in almost the same decrease (~85%) as α7Δ428-432.The results suggest that the upper part of the MA helix plays a more important role in binding PICK1 than the lower part of the MA helix.
Figure S3.Overlay of 2D saturation transfer (STD) NMR spectra of the α7nAChR TMD+ICD without (purple) and with (green) saturation of the PICK1 I33 amide proton (~11.6 ppm).STD spectra were collected in an interleaved fashion with off-and on-resonance saturation of 1 HN peak (~11.6 ppm) of I33 for 0.5s saturation and a recycle time of 1.5s.The off-resonance frequency was set at 20 ppm, which is far away from the 1 H frequencies of both the 15 N-labeled α7nAChR TMD+ICD and the unlabeled PICK1 PDZ domain.The selective saturation was achieved using an IBURP2 pulse train (50 ms Gaus1.1000-shaped with an inter-pulse delay of 4 μs).The spectra were acquired at 800 MHz NMR spectrometer at 45 ºC.

Figure S4 .
Figure S4.Inter-protein paramagnetic relaxation enhancement (PRE) NMR.2D 1 H-15 N TROSY-HSQC NMR spectra of the α7nAChR TMD+ICD were collected in the presence of PICK1 PDZ domain in the paramagnetic (orange) and diamagnetic (blue) conditions, respectively.The paramagnetic condition resulted from the MTSL labeling at residues C44 and C46 of the PDZ.The diamagnetic condition was introduced by ascorbic acid (2.5 mM).Several inserts are provided to assist visualization of signal changes under the paramagnetic and diamagnetic conditions.These spectra were acquired at 800 MHz NMR spectrometer at 35 ºC.

Figure
Figure S5.FSC231 inhibition of PICK1 pulldown by α7nAChR.(a) PICK1 was pulled down by α7nAChR conjugated bungarotoxin resin (BGT-α7), but not resin alone (BGT).(b) The PICK1 inhibitor FSC231 inhibited PICK1 binding to BGT-α7 in a concentration dependent manner.All samples were washed five times in PBS with 0.1% Triton X100, resolved by SDS-PAGE, and stained with Sypro Ruby.

FSC231
Figure S6.Representative interactions between α7nAChR ICD (colored in yellow or orange, residues are presented in VDW and label in black) and PICK1 PDZ (colored in cyan, residues are presented in licorice and labeled in cyan).(a) Interactions between the α7nAChR MA helix and PICK1 βB that lines the canonical binding grove of the PDZ domain.(b) Interactions between the α7nAChR MX helix and loop residues prior the PICK1 βB.(c) α7nAChR ICD residues at the lower MA helix (E416) contacts with the PICK1 loop residue Q42.(d) α7nAChR residues in the ICD flexible loop interact with PICK1 residues prior (K81) and on (Q91) αB helix.Note the salt bridge between D371 and K81.(e) and (f) Residues of the PICK1 PDZ domain interact with residues not only on the principal side (yellow) but also complementary side (orange) of α7nAChR subunits.

Figure S7 .
Figure S7.Impact of mutations in the MA helix of α7nAChR to binding the PICK1 PDZ domain.(a) Sequence alignment of MA helices of the human α7, α4, and β2 nAChRs.The α7 residues colored in orange are closely interacting with the PDZ domain in the α7nAChR-PICK1 complex structural model.To validate the complex structure and assess how mutations in the α7 MA helix influence the α7nAChR-PICK1 complex formation, we designed an α7 mutant of three mutations (N423D, E430D and S431D), named α7DDD, to match these three α7 residues with the equivalent residues in α4 or/and b2, because neither α4 nor b2 was found binding PICK1 previously. 1 (b) Overlay of size exclusive chromatography (SEC) profiles of the purified wild type (α7WT, black) and mutant (α7DDD, red) α7nAChR.The same SEC profile and peak position (~1.4 ml) of α7WT and α7DDD suggests that α7DDD retains the pentameric assembly as α7WT.The SEC was performed using a Superdex 200 Increase 3.2/300 column with 10 mM phosphate buffer at pH 7.8, 150 mM NaCl and 0.05% LDAO.(c) 15% SDS-PAGE gel showing pulldown of the PICK1 PDZ domain by α7WT and α7DDD with their his-tags as baits.Comparing to α7WT, α7DDD shows a ~50% decrease in pulldown of PICK1 based on band intensity ratios of PICK1/α7WT vs. PICK1/α7DDD.The slower migration of α7DDD in the SDS-PAGE results from an increase of the net negative charge in α7DDD as reported previously. 2 The experiment was carried out following the protocol described in the Method section.